Detection of Humoral Response Using a Recombinant Heat Shock Protein 70, DnaK, ofMycoplasma haemofelisin Experimentally and Naturally Hemoplasma-Infected Cats

Abstract

Hemoplasmas is the trivial name given to a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. Of the feline hemoplasmas, Mycoplasma haemofelis is the most pathogenic, while "Candidatus Mycoplasma haemominutum" and "Candidatus Mycoplasma turicensis" are less pathogenic. Shotgun libraries of fragmented M. haemofelis genomic DNA were constructed, and random colonies were selected for DNA sequencing. In silico-translated amino acid sequences of putative open reading frames were compared to mass spectrometry data from M. haemofelis protein spots identified as being immunogenic by two-dimensional gel electrophoresis and Western blotting. Three of the spots matched the predicted sequences of a heat shock protein 70 (DnaK) homolog, elongation factor Ts, and a fragment of phosphoglycerate kinase found during library screening. A full-length copy of the M. haemofelis dnaK gene was cloned into Escherichia coli and recombinantly expressed. Recombinant M. haemofelis DnaK was purified and then used in Western blotting and an enzyme-linked immunosorbent assay (ELISA) to investigate the humoral immune response during acute infection in cats experimentally infected with M. haemofelis, "Ca. Mycoplasma haemominutum," or "Ca. Mycoplasma turicensis". The recombinant M. haemofelis DnaK ELISA also was used to screen clinical samples submitted for hemoplasma PCR testing to a commercial laboratory (n ‫؍‬ 254). Experimentally infected cats became seropositive following infection, with a greater and earlier antibody response seen in cats inoculated with M. haemofelis than those seen in cats inoculated with "Ca. Mycoplasma haemominutum" or "Ca. Mycoplasma turicensis," by both Western blotting and ELISA. Of the clinical samples, 31.1% had antibodies detected by the ELISA but only 9.8% were positive by PCR for one or more hemoplasmas.

Quantification of the humoral immune response and hemoplasma blood and tissue loads in cats coinfected with ‘Candidatus Mycoplasma haemominutum’ and feline leukemia virus

“…Seroconversion was detected in all seven CMhm-infected cats but not in the cat that had remained CMhm PCR-negative, thereby confirming and extending the results of a recently published study [12]. The ELISA signal-to-noise ratios of most CMhm-infected cats were lower than those reported for a cat experimentally infected with Mhf using the same method [11].…”

“…The first assessment of the antibody response in cats experimentally infected with CMhm used whole CMhm antigen on blood smears of infected cats to detect anti-CMhm serum antibodies [10]. Most recently, two serological assays, both enzyme-linked immunosorbent assays (ELISAs), were developed for the analysis of the humoral immune response in cats experimentally infected with hemoplasma [11,12]. The assays make use of the recombinant heat shock protein 70, DnaK, of Mhf, and have been applied successfully to detect anti-DnaK antibodies in cats experimentally infected with hemoplasmas and to quantify the humoral immune response to Mhf [11,12].…”

“…Most recently, two serological assays, both enzyme-linked immunosorbent assays (ELISAs), were developed for the analysis of the humoral immune response in cats experimentally infected with hemoplasma [11,12]. The assays make use of the recombinant heat shock protein 70, DnaK, of Mhf, and have been applied successfully to detect anti-DnaK antibodies in cats experimentally infected with hemoplasmas and to quantify the humoral immune response to Mhf [11,12]. Thus far, however, the humoral immune response of cats to CMhm has never been quantitatively assessed by ELISA over time, although this information would provide essential insight into the poorly understood pathogenesis of CMhm infections in felines.…”

Molecular characterization of the uncultivatable hemotropic bacterium Mycoplasma haemofelis

“…Of particular interest is the 25-29 kDa region, which contains a number of immunogenic proteins identified as distinct spots on a 2D immunoblot [7], and as a variable band on one dimensional - polyacrylamide gel electrophoresis [9]. Unfortunately the majority of immunogenic spots in this region did not correspond to visible spots on gels.…”

Genome Sequence for “Candidatus Mycoplasma haemominutum,” a Low-Pathogenicity Hemoplasma Species

“…Mycoplasma haemominutum" strain Birmingham 1 was purified from blood taken from an experimentally infected specific-pathogen-free cat at high parasitemia, as described previously (2). This low-passage strain, derived from a naturally infected cat (15), has been used in previous infection kinetics studies (9,14,16,17).…”